Scientia Agricultura Sinica

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Scientia Agricultura Sinica. 2022, 55(10): 0. doi:10.3864/j.issn.0578-1752.2022-10-0

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Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum

LIU RuiDa, GE ChangWei, WANG MinXuan, SHEN YanHui, LI PengZhen, CUI ZiQian, LIU RuiHua, SHEN Qian, ZHANG SiPing, LIU ShaoDong, MA HuiJuan, CHEN Jing, ZHANG GuiYin, PANG ChaoYou

Scientia Agricultura Sinica. 2022, 55(10): 1877-1890. doi:10.3864/j.issn.0578-1752.2022.10.001

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【Objective】As one of the largest transcription factor families in plants, MYB genes play an important role in resisting stress. The MYB transcription factor GhMYB108 was cloned and analyzed to verify its role in drought stress response, which laid a foundation for further study on the molecular mechanism of GhMYB108 regulating drought tolerance in G. hirsutum.【Method】Through the analysis of unpublished drought transcriptome data, GhMYB108 was identified to drought response. The target gene was amplified from the root cDNA by polymerase chain reaction (PCR). Through bioinformatics analysis of gene structure characteristics, the sequence information and phylogenetic relationship of these genes were predicted. The obtained gene promoter sequences were analyzed by Plant Care website. The genes expression characteristics under different stress conditions were analyzed using Real time fluorescence quantitative PCR (qRT-PCR). The location of GhMYB108 protein was determined by subcellular localization. The transcriptional activity was tested in yeast cell; The GhMYB108 gene was silenced using Virus induced gene silencing (VIGS), and the gene silencing efficiency was detected by qRT-PCR. The phenotypic changes before and after drought treatment were observed and the survival rate was counted. The relevant physiological and biochemical indexes were measured by Solarbio Kit; The relationship between GhMYB108 and ABA was analyzed by spraying ABA and Fluridone on cotton leaves.【Result】GhMYB108 (Gh_A10G1563) was cloned from G. hirsutum, with 879 bp length and 292 amino acids. Its protein relative molecular weight and isoelectric point is 33.288 kD and 6.037, respectively. Multiple sequence alignment and conserved domain analysis showed that GhMYB108 contains two highly conserved MYB binding domains, which belongs to a typical R2R3 MYB transcription factor. Phylogenetic analysis of different species showed that GhMYB108 was highly homology with ATMYB108, ATMYB78 and ATMYB2, belonging to the same subfamily. Previous studies found that ATMYB108, ATMYB78 and ATMYB2 were related to drought and ABA signaling pathway. GhMYB108 located in the nucleus and had transcriptional activation activity. The expression level of GhMYB108 was the highest in roots and the lowest in stems, and was induced by abiotic stresses including natural drought, 18% PEG 6000 simulated drought, salt stress and low temperature. The GhMYB108 silenced plants showed a critical phenotype under natural drought conditions. Compared with the control, the silenced plants showed more serious wilting and decreased survival rate. Some physiological and biochemical indexes also changed significantly, such as accelerated leaf water loss rate, increased malondialdehyde content, decreased leaf relative water content and proline content, and decreased CAT and POD activities. Through DAB and NBT staining, the hydrogen peroxide (H₂O₂) and superoxide anion (O₂^-) were significantly accumulated in plants. By spraying the hormone ABA or Fluridone on cotton leaves, we found that GhMYB108 could be positively regulated by ABA signal.【Conclusion】GhMYB108 positively regulates cotton drought resistance and is positively regulated by ABA signal.

Screening of Sweetpotato Varieties Tolerant to Low Nitrogen at Seedling Stage and Evaluation of Different Nitrogen Efficiencies

FAN WenJing,LIU Ming,ZHAO Peng,ZHANG QiangQiang,WU DeXiang,GUO PengYu,ZHU XiaoYa,JIN Rong,ZHANG AiJun,TANG ZhongHou

Scientia Agricultura Sinica. 2022, 55(10): 1891-1902. doi:10.3864/j.issn.0578-1752.2022.10.002

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【Objective】The objective of this article was to establish a low-N tolerance evaluation system for sweetpotato varieties, to screen low-N-tolerant genotypes and evaluate different N efficiency categories and to provide a theoretical basis for studying the low-N-tolerant physiological mechanism of sweetpotato varieties and mining N-efficient genes.【Method】Under the treatment of low N stress (0 mmol·L^-1) and normal N application (14 mmol·L^-1) of hydroponic experiment. Selected 126 sweetpotato varieties from different sweetpotato areas at home and abroad, we collected eleven indicators, including the shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, root-to-shoot ratio, vine length, root length, leaf number, CCI, nitrogen accumulation, nitrogen physiological utilization efficiency, and calculated the low-N tolerance index of all indicators. The study carried out principal component analysis by using the comprehensive membership function method, regression analysis and cluster analysis to comprehensively evaluate the low-N-tolerant sweetpotato varieties and N efficiency types.【Result】1) Under low N level, the average of the shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, vine length, root length, leaf number, CCI, and N accumulation of 126 tested sweetpotato varieties was lower than that under normal N level, while the average of root-to-shoot ratio and nitrogen physiological utilization efficiency were higher than that under normal N level; 2) The variation coefficient of shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, root-to-shoot ratio, vine length, root length, leaf number, nitrogen accumulation, nitrogen physiological utilization efficiency at low-N stress was higher than that at normal N level, and the amplification of them were ranked as shoot biomass increase>plant biomass increase ratio>root biomass increase>leaf number>shoot biomass>nitrogen physiological utilization efficiency>nitrogen accumulation>root length> root-to-shoot ratio>Vine length; 3) The principal component analysis was carried out on the low-N-tolerant index of eleven indicators, extracted three principal components, the cumulative variance contribution rate of which was 72.67%, and calculated the comprehensive evaluation Y-value; 4) The correlation between the low-N-tolerant index of shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, leaf number, vine length, root length, root-to-shoot ratio, nitrogen accumulation, nitrogen physiological utilization efficiency and the Y-value were highly significant(P<0.01), among them, the correlation of shoot biomass increase, root biomass increase, plant biomass increase ratio, nitrogen accumulation and shoot biomass was higher, with the correlation coefficients of 0.85, 0.86, 0.81, 0.79 and 0.73, respectively; 5) The regression equation of the Y-value screened eight indicators to evaluate low-N tolerance of sweetpotato varieties, and the cluster analysis on eight indicators showed that the sweetpotato genotypes were classified into three types, low-N-tolerant, intermediate, and low-N-sensitive. The agronomic traits and N efficiency traits of three sweetpotato categories were analyzed by variance analysis.【Conclusion】Shoot biomass, shoot biomass increase, root biomass increase, root length, vine length, leaf number, nitrogen accumulation and nitrogen physiological utilization efficiency were selected as the low-N tolerance evaluation indicators of sweetpotato; The study screened 7 low-N-tolerant sweetpotato varieties: 13104-2/Zishu1, Yibinhongxinshu, Zhezishu 2, Yuzi 3, Yuzi 6, Luozi 1 and Yuzixiang 10; The results of variance analysis showed that the low-N-tolerant varieties performed better than the intermediate and the low-N sensitive varieties, and there are significant difference in five traits: shoot biomass, shoot biomass increase, root biomass increase, vine length and nitrogen accumulation.

Effects of Interspecific Distances on Soil Physicochemical Properties and Root Spatial Distribution of Maize-Soybean Relay Strip Intercropping System

REN JunBo, YANG XueLi, CHEN Ping, DU Qing, PENG XiHong, ZHENG BenChuan, YONG TaiWen, YANG WenYu

Scientia Agricultura Sinica. 2022, 55(10): 1903-1916. doi:10.3864/j.issn.0578-1752.2022.10.003

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【Objective】The purpose of this study was to explore the effects of interspecific distance on soil environment and root spatial distribution of maize-soybean intercropping, so as to provide a theoretical basis for crop roots to regulate the efficient utilization of nutrients.【Method】The field experiments were used one-factor randomized block design with 5 root interaction modes: maize-soybean intercropping spacing 30 cm (MS30), 45 cm (MS45), 60 cm (MS60), maize monoculture row spacing 100 cm (MM100), and soybean monoculture row spacing 100 cm (SS100). The changes of soil oxygen content, soil respiration rate, soil nutrient content, soil aggregate and root distribution were investigated.【Result】From dough stage (R4) to maturity stage (R6) of maize, as well as from the beginning seed (R5) to full maturity (R8) of soybean, the daily average soil oxygen content and soil respiration rate of intercropping treatment initially increased and then decreased later with the increase of interspecific distance; The soil oxygen content of maize was the highest in MS45, the lowest under MS30, while the soil respiration rate of intercropping was significantly lower than the monoculture. The soil respiration rate of soybean was the highest under MS45, which was 130.00% higher than that under SS100, while the soil oxygen content of intercropping was lower than that of monoculture. Compared with monoculture, the content of water-stable aggregates >5 mm in the soil of intercropping maize, the content of water-stable aggregates of 5-2 mm in the soil of intercropping soybean and the soil NO- 3-N were significantly increased, by 19.26%, 4.49%, and 18.07%, respectively; Among which, those contens under MS45 was the highest. During the co-growing period, compared with monoculture, the spatial distribution of maize and soybean roots under each intercropping treatment was asymmetrical, and the intercropping maize roots could extend horizontally below the space of soybean rows and grow deeper vertically. The root system of intercropping soybean was obviously inclined to the growth of soybean belt, and the total root length, root surface area, root volume and root dry weight of intercropping maize and soybean were lower than that of monoculture. After the maize was harvested, the intercropping soybean root system resumed growth and further extended in the horizontal and vertical directions. The root volume of MS45 was higher than those of monoculture. PCA analysis showed that soil nutrient content and water stable aggregate index were positively correlated with root morphological parameters.【Conclusion】Reasonable interspecific distance promoted the formation of soil large aggregates, increased soil oxygen content, improved soil aeration environment and soil nutrient, optimized the spatial distribution of crop roots, and promoted root growth and development.

Adaptability Evaluation of Staple Crops Under Different Precipitation Year Types in Four Ecological Regions of Inner Mongolia Based on APSIM

LIU XiaXia, LI Yang, WANG Jing, HUANG MingXia, BAI Rui, SONG Yang, HU Qi, ZHANG JiaYing, CHEN RenWei

Scientia Agricultura Sinica. 2022, 55(10): 1917-1937. doi:10.3864/j.issn.0578-1752.2022.10.004

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【Objective】 In order to provide the important scientific reference for optimizing the layout of staple crops in Inner Mongolia, the adaptability of staple crops (maize, potato, oats, canola, oil and edible sunflower) was evaluated in four ecological regions of Inner Mongolia under different precipitation year types.【Method】Four typical sites in four ecological regions were selected. The validated APSIM model was used to quantify the potential yields, rainfed yields and yield gaps of six crops. Yield reduction rates of rainfed yields relative to potential yields under different precipitation year types were calculated to evaluate the adaptability of staple crops. Crop water production functions were conducted to analyze crop water sensitivity. 【Result】RMSE between simulated and observed vegetative growth period, reproductive growth period, dry yield was 10.1 d, 8.9 d, and 1 322.4 kg·hm^-2, respectively. NRMSE between observed and simulated vegetative growth period, reproductive growth period, and dry yield was 14.6%, 19.2%, and 22.6%, respectively. The validation results showed that APSIM could effectively simulate the growth, development, and yield of each crop in different regions. The potential dry yields of maize, potato, oats, canola, oil sunflower, and edible sunflower were 12 024±4 874, 7 315±806, 6 611±906, 2 424±326, 2 721±205, and 4 905±428 kg·hm^-2, respectively. The potential yields of oats and edible sunflower reached the maximum values in the north foot of Yinshan Mountains while potential yields of other four crops reached the maximum values in the Loess Plateau. The rainfed dry yields of maize, potato, oats, canola, oil sunflower, and edible sunflower were 3 056±2 902, 3 337±1 608, 2 974±1 677, 912±511, 869±618, and 1 508±984 kg·hm^-2, respectively. Average rainfed yields of the six crops increased from west to east and reached the maximum values in Da Hinggan Mountains. Yield gaps of maize, potato, oats, canola, oil sunflower, and edible sunflower were 8 968±5 844, 3 978±2 358, 3 637± 2 122, 1 512±832, 1 852±749, and 3 397±1 328 kg·hm^-2, respectively. Except maize and oats, the yield gaps of four crops decreased from west to east and reached the minimum values in Da Hinggan Mountains. Taking the yield reduction rate from potential to rainfed conditions as the drought index under the rainfed condition and considering the variation coefficients of rainfed yields, it was not suitable to plant crops in any years in the Loess Plateau. In the north foot of Yinshan Mountains, crops were not suitable for planting in dry years. Potato was suitable for planting in normal years, while potato and oats were suitable for planting in wet years. At the foothills of Yanshan hilly area, crops were not suitable for planting in dry years. Potato and oats were suitable for planting in normal years while six crops were all suitable for planting in wet years. In Da Hinggan Mountains, potato, oats, canola, and edible sunflower were suitable for planting in dry years, while six crops were all suitable for planting in normal and wet years. The linear correlations between the relative evapotranspiration and the relative yield of the six crops were all significant (P<0.01) with R² ranging from 0.84 to 0.99. The sensitivity of crop to water stress was in the order of oil sunflower, edible sunflower, maize, oats, canola, and potato.【Conclusion】This study revealed the adaptability of staple crops under different precipitation year types in the four ecological regions of Inner Mongolia. There was a large difference in water sensitivity of six crops. Under rainfed condition, potato were suitable for planting in normal and wet years in the north foot of Yinshan Mountains and the foothills of Yanshan hilly area, and in all year types in Da Hinggan Mountains. Oats were suitable for planting in wet years in the north foot of Yinshan Mountains, in normal and wet years in the foothills of Yanshan hilly area and in all year types in Da Hinggan Mountains. Canola and edible sunflower were suitable for planting only in wet years in the foothills of Yanshan hilly area and in all year types in Da Hinggan Mountains. Maize and oil sunflower were suitable for planting only in wet years in the foothills of Yanshan hilly area and in normal and wet years in Da Hinggan Mountains.

Establishment and Application of Rapid Quantitative Detection of Viable Plasmodiophora brassicae by PMAxx-qPCR Method

LI XiaoJing,ZHANG SiYu,LIU Di,YUAN XiaoWei,LI XingSheng,SHI YanXia,XIE XueWen,LI Lei,FAN TengFei,LI BaoJu,CHAI ALi

Scientia Agricultura Sinica. 2022, 55(10): 1938-1948. doi:10.3864/j.issn.0578-1752.2022.10.005

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【Objective】Plasmodiophora brassicae is an obligate endoparasite that causes clubroot disease, which is the most devastating soil-borne disease in brassica crops. The propidium monoazide xx (PMAxx) could selectively bind to the chromosomal DNA of dead spores and therefore block DNA amplification by real-time fluorescent quantitative PCR (qPCR). In the present study, a strategy involving a PMAxx pre-treatment followed by the qPCR (PMAxx-qPCR) assay was developed for quantifying viable spores of P. brassicae, so as to provide a basis for early detection and prevention measurement of cloobroot disease. 【Method】 PMA and PMAxx with concentrations of 0, 5, 10, 20, 40 and 60 µmol·L^-1 were prepared, respectively, and were used to pre-treat P. brassicae prior to DNA extraction, followed by qPCR. The inhibitory effects of PMA and PMAxx on DNA amplification of P. brassicae dead spores were compared, and the optimal nucleic acid dye and concentration to distinguish between live and dead spores were determined. The illumination time was set as 0, 2, 5, 10, 15 and 20 min, respectively, and the optimal exposure time was optimized to establish a PMAxx-qPCR assay for selectively detection of viable spores of P. brassicae. The mixed suspensions with different ratios of dead and viable spores (0, 0.01%, 0.1%, 1%, 10%, 25%, 50%, 75% and 100% viable spores) were prepared to determine the suitability of PMAxx-qPCR assay for distinguishing viable and dead spores. The assay was also applied to quantitative detection of viable spores of P. brassicae in 25 field soil samples. 【Result】 PMAxx showed a better discrimination effect than PMA on the viable and dead spores of P. brassicae. When the concentration of P. brassicae was 1×10⁸spores/mL, the optimal PMAxx concentration and light exposure time were 4 μmol·L^-1 and 10 min, respectively. The amplification of dead spores could be inhibited effectively, and only the DNA of living spores was targeted for selective amplification. For pre-defined ratio of viable spores, there was a good linear relationship between the lg of the P. brassicae DNA concentration assessed by PMAxx-qPCR and the theoretical viability (R²=0.992). For soil samples, viable P. brassicae was quantified in 11 of 25 samples, with infestation levels of approximately 32.35-6.97×10³ fg·g^-1. 【Conclusion】 The established method could quantitatively detect the viable spores of P. brassicae, with advantages of rapid, efficiency and sensitivity, which could be useful for avoiding the inability of qPCR method to distinguish between viable and nonviable spores. Application of the assay may potentially improve P. brassicae control and disease management.

Migration Pathway of Spodoptera frugiperda in Northwestern China

WU QiuLin,JIANG YuYing,LIU Yuan,LIU Jie,MA Jing,HU Gao,YANG MingJin,WU KongMing

Scientia Agricultura Sinica. 2022, 55(10): 1949-1960. doi:10.3864/j.issn.0578-1752.2022.10.006

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【Background】As a major migratory insect pest putting the whole world on alert, the fall armyworm Spodoptera frugiperda warned by Food and Agriculture Organization of the United Nations (FAO) poses a serious threat to the agriculture production (including maize) of China from the end of 2018. Taking advantages of the long-distance transport of seasonal monsoons and its self-powered migratory capacity, S. frugiperda performs two migration pathways to fly across the eastern and western China separately, which has caused regional dispersal and severe infestation. According to 2-year systematic investigation in China, the west migration route of S. frugiperda ends in Northwestern China, especially in Ningxia and Alxa Left Banner of Inner Mongolia. However, little is known about the source areas of S. frugiperda population invading Northwestern China, and few reports explore the migration route of this devastating pest through the whole western China.【Objective】The objective of this study is to accurately analyze on the key atmospheric factors driving the immigration of S. frugiperda into the Northwestern China, source regions of the first populations to arrive, and Asian monsoon-induced migration pathways of S. frugiperda, which can provide fundamental evidence for the early warning and regional management and control of this invasive pest in China.【Method】Based on the invasion dynamics of S. frugiperda in Ningxia of the Northwestern China and meteorological data, a meso-scale numerical model, insect’s flight trajectory calculating program, and Geographic Information System (GIS) were used to identify the atmospheric transport backgrounds, simulate the succussive 1-3 night migration routes and trace their source regions of S. frugiperda in the Northwestern China.【Result】The southerly summer monsoon from July to September each year was the key factor for the successive and successful immigration of S. frugiperda into Ningxia and other regions of Northwestern China, of which their major source populations of S. frugiperda were located in southeastern Gansu and eastern Sichuan, while some were from western Shaanxi. In addition, southwestern Chongqing, northeastern Yunnan and part of western Shanxi could possibly provide population source of S. frugiperda.【Conclusion】Under the influence of southerly Asian summer monsoons, S. frugiperda can fly towards the north for 1-3 successive nights via its west migration pathway “Yunnan-Sichuan and Chongqing-Shaanxi and Gansu-Ningxia” in China, which originates from Myanmar and ends in Inner Mongolia, China. In particular, the government should be vigilant against the occurrence and damage of this devastating pest in the maize-cropping regions of Northwestern China while the preponderance of early southerly wind is advanced and wind speed gets strong during July to September.

Spatial Distribution Pattern and Sampling Technique of Conogethes punctiferalis Larvae in Maize Fields

LI ShaoHua,WANG YunPeng,WANG RongCheng,YIN Ping,LI XiangDong,ZHENG FangQiang

Scientia Agricultura Sinica. 2022, 55(10): 1961-1970. doi:10.3864/j.issn.0578-1752.2022.10.007

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【Objective】The yellow peach moth, Conogethes punctiferalis, as an agricultural insect pest, its damage to maize ears has become more and more serious in Huang-Huai-Hai maize-producing areas of China in recent years, threatening the safe production of maize and the food safety. The spatial distribution pattern is an important ecological attribute of insect population, the objective of this study is to research the spatial distribution pattern of C. punctiferalis larvae in maize fields, clarify the spatial distribution characteristics of the pest, and to provide scientific bases for formulating field sampling program of C. punctiferalis larvae in maize fields, forecasting and effective management of the insect pest on maize.【Method】The spatial distribution pattern of the population of C. punctiferalis larvae in maize fields was studied by traditional statistical method (aggregation indexes, Taylor’s power law and Iwao’s regression model) and geostatistical method. Based on the Iwao’s regression model, the theoretical sampling number of C. punctiferalis larvae in fields was determined, and the maximum theoretical sampling number with different admissible errors (D=0.1, 0.2, 0.3) and the putative economic thresholds (m₀=0.5, 1, 1.5, 2 larvae per plant) was also determined by the sequential sampling.【Result】The results of the two kinds of statistical methods showed that the spatial distribution pattern of C. punctiferalis larvae belonged to aggregation distribution. The analysis of some aggregation indexes showed that spatial distribution pattern of C. punctiferalis larvae belonged to aggregation distribution. The results of Taylor’s power law showed that the spatial distribution pattern of C. punctiferalis larvae belonged to aggregation distribution, and the aggregation intensity increased with the population density. The Iwao’s regression model proved that the spatial distribution pattern of C. punctiferalis larvae belonged to negative binomial distribution in aggregation distributions. The parameters of semivariogram models indicated that the optimal fitting models of C. punctiferalis larvae were the spherical, exponential and linear models. The three-dimensional and two-dimensional maps from Kriging interpolations showed that the aggregation centers of C. punctiferalis larvae were located at the edges of the fields. Based on sampling technique from the Iwao’s regression model, the theoretical sampling number of C. punctiferalis larvae in maize fields was determined when the confidence probability t=2 and different mean densities m=0.5, 1, 2, 3, 4, 5, 10 and 15. The maximum theoretical sampling number was also determined by the sequential sampling. Assuming t=2, D=0.1, 0.2, 0.3, when m₀=0.5 larva per plant, the maximum theoretical sampling numbers were 3 417, 854 and 380, respectively; when m₀=1 larva per plant, the maximum theoretical sampling numbers were 1 717, 429 and 191, respectively; when m₀=1.5 larvae per plant, the maximum theoretical sampling numbers were 1 150, 287 and 128, respectively; when m₀=2 larvae per plant, the maximum theoretical sampling numbers were 867, 217 and 96, respectively.【Conclusion】The spatial distribution pattern of C. punctiferalis larvae belongs to the negative binomial distribution in aggregation distributions, and the aggregation centers were located at the edges of the fields. The maximum theoretical sampling number based on the sequential sampling in maize fields can be used for monitoring and management of C. punctiferalis larvae.

Effects of Basal Nitrogen and Foliar Zinc Application at the Early Filling Stage on Zinc Enrichment and Protein Components Content in Wheat Grain

WU TianQi,LI YaFei,SHI JiangLan,NING Peng,TIAN XiaoHong

Scientia Agricultura Sinica. 2022, 55(10): 1971-1986. doi:10.3864/j.issn.0578-1752.2022.10.008

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【Objective】 Foliar zinc (Zn) application is an effective agronomic biofortification strategy to realize Zn enrichment of wheat grains and to combat the human Zn malnutrition. The aim of this study was to investigate the effects of spraying Zn with different nitrogen (N) inputs on Zn enrichment and content of protein and protein components in wheat whole grain and flour.【Method】 Based on the long-term positioning experiment, the spraying experiment of Zn in wheat for two consecutive seasons was conducted during 2018-2020. A split plot design was used with soil N rates of 0 (N₀), 120 (N₁₂₀) and 240 ∙hm^-2 (N₂₄₀) as the main plot factor, and the foliar application of distilled water (Zn₀) and 0.4% ZnSO₄·7H₂O (Zn₁) as subplot. The indexes were analyzed for this study, including Zn content of various nutritional organs, Zn mobilization and distribution from leaf and other vegetative organs to grain, and protein and protein component content in grains and flour at the early filling stage and maturation stage. 【Result】Compared with N₀, the grain yield under N₁₂₀ and N₂₄₀treatments was significantly increased by 88%-114%, but there was no significant difference between N₁₂₀ and N₂₄₀. Regardless of the N inputs, the foliar Zn application could significantly increase the Zn concentration in grains and flour and the grain Zn content reached the biofortification standard. Among those treatments, the Zn concentration of wheat grains under N₁₂₀ and N₂₄₀ was increased by 0.95 and 1.12 times than that under N₀, respectively. Compared with N₀, the N inputs increased the translocation of N and Zn transferred from leaf and other vegetative organs to grain at early grain filling stage, but reduced the transfer ratio of N and Zn: N decreased from 60.2% to 48.6% and Zn decreased from 42.3% to 26.5%. A significant positive linear correlation was found between the amount of N and Zn mobilization and the content of N and Zn in grain at maturity, and the synergistic effect of N and Zn was more significant at Zn₁. Compared with the early filling stage, the content of storage protein (gliadin and glutenin) in grains and flour at the mature stage increased significantly, accounting for about 80%-84% of the protein content. The content of gliadin and glutenin in grain and flour was increased by N application more than that of albumin and globulin, and the gluten was the largest. The content of protein and protein components in grain and flour were not affected by spraying Zn. However, in terms of Zn₁, the increase of gluten content in grain and flour was higher than that under the condition of Zn₀with the increase of N dosage, which was 37.5% and 38.1%, respectively.【Conclusion】Foliar Zn application could achieve Zn-rich grains but did not affect the content of protein and protein components in grains and flour, indicating that there was sufficient protein pool for Zn storage in grains and flour. Therefore, a reasonable amount of soil N combined with foliar Zn application could increase the N, Zn content and nutritional quality of the grains by ensuring high and stable yield on potentially Zn-deficient calcareous soils.

Spatial-Temporal Variability of Soil Nutrients and Assessment of Soil Fertility in Erhai Lake Basin

GUO YingXin,CHEN YongLiang,MIAO Qi,FAN ZhiYong,SUN JunWei,CUI ZhenLing,LI JunYing

Scientia Agricultura Sinica. 2022, 55(10): 1987-1999. doi:10.3864/j.issn.0578-1752.2022.10.009

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【Objective】By studying the spatial-temporal variation characteristics of tobacco-planting soil nutrients in Erhai Lake Basin (ELB), the objective of grading evaluation and spatial visualization of tobacco-planting soil fertility in this region was achieved, so as to provide a scientific basis for the nutrient management, balanced fertilization, and the control of agricultural non-point source pollution of tobacco-planting areas in ELB.【Method】Based on the 964 tobacco-planting soil samples in ELB collected in 2011-2013, 2018 and 2020, this study explored the spatial-temporal variability of nutrients and regional distribution patterns by using Geostatistics, Geographic Information Systems (GIS) technology, and Fuzzy integrated fertility index method to quantify the soil fertility in tobacco-planting areas. 【Result】The average values of soil pH, soil organic matter (SOM), total nitrogen (TN), Olsen-P (AP) and available potassium (AK) of tobacco-planting soil in ELB were 7.3, 59.6 g·kg^-1, 3.5 g·kg^-1, 54.4 mg·kg^-1, and 192.0 mg·kg^-1, respectively, all of which belonging to moderate variation. Tobacco-planting soil was rich in SOM, TN, AP and AK, and the proportions of areas within the upper-middle level accounted for 85.2%, 93.8%, 94.5% and 78.8%, respectively, showing obvious variation at regional scale. The area of tobacco-planting soil fertility were graded to five levels (from high to low: I to V), which accounted for 8.4%, 25.0%, 40.3%, 23.3% and 3.0%, respectively. The pH of tobacco-planting soil was relatively alkaline in ELB, which was higher in the northern than in the southern; the highest concentrations of SOM and TN occurred in the northern and the western region; the areas with high AP concentration were distributed in patches in the northern, eastern and western region of Erhai Lake; the areas with high AK concentration were distributed in flakes in the northern and eastern of Erhai Lake. 【Conclusion】Collectively, the fertility of the tobacco-growing soil in ELB was in high level, and the high-quality soil areas above grade III were mainly distributed in the northern and eastern region. Meanwhile, the tobacco-planting soil in the northern and western Erhai Lake were rich in nitrogen and phosphorus, and there was a risk of agricultural non-point source pollution in the region.

Effects of Arbuscular Mycorrhizal Fungi on Soil N₂O Emissions During Maize Growth Periods

ZHANG XueLin,HE TangQing,ZHANG ChenXi,TIAN MingHui,LI XiaoLi,WU Mei,ZHOU YaNan,HAO XiaoFeng

Scientia Agricultura Sinica. 2022, 55(10): 2000-2012. doi:10.3864/j.issn.0578-1752.2022.10.010

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【Objective】 The aim of this study was to understand the mechanism of arbuscular mycorrhizal fungi (AMF) on soil nitrous oxide (N₂O) emissions, so as to provide the theoretical basis for increasing maize yield, improving nitrogen (N) use efficiency and reducing greenhouse gas emissions. 【Method】 A 2-factorial greenhouse experiment was established during maize growth periods in 2016 and 2017. The factors were as follows: (1) N fertilizer rates (180 kg N·hm^-2 (N1) and 360 kg N·hm^-2 (N2)), and (2) three mycorrhizae treatments, including a control (M0, neither roots nor AMF could enter the hyphal chamber from the growth chamber), an AMF treatment (M1, only AMF can enter the hyphal chamber from the growth chamber), and a root treatment (M2, both roots and AMF can enter the hyphal chamber from the growth chamber). Maize grain yield, plant biomass and their N accumulation, and soil N₂O flux were measured. Soil bacterial community structure and diversity at maize maturity stage was determined by using the high throughput sequencing technique on Hiseq 2500 PE250. 【Result】 Both N fertilizer rates and mycorrhizae treatments significantly affected maize yield, plant N accumulation and soil N₂O flux. Compared with M0, maize yield under M1 and M2 under the conditions of N1 input increased by 38% and 82%, by 30% and 52% for aboveground N accumulation, respectively, and reduced by 26% and 65% for soil inorganic N, respectively. However, under the conditions of N2 input, the maize yield under M1 and M2 increased by 16% and 48%, by 9% and 33% for aboveground N accumulation, and reduced by 34% and 55% for soil inorganic N, respectively. Compared with the M0, the total N₂O emission of M1 and M2 treatments reduced by 17% and 40% under the conditions of N1 input, and by 41% and 67% for the N₂O emission intensity, respectively; while under the conditions of N2 input, the total N₂O emission reduced by 26% and 45%, and by 28% and 57% for the N₂O emission intensity, respectively. Nonmetric multidimensional scaling analysis showed that both N fertilizer rates and mycorrhizae treatments had significant effects on bacterial communities’ composition. Compared with N1, the relative abundance of Proteobacteria and Gemmatimonadetes under N2 treatment on phyla level reduced by 6% and 15%, increased by 32% for Actinobacteria, while on genera level, the Streptomyces increased by 27%, and reduced by 8% for Gemmatimonas. Compared with M0 under the conditions of N1 input, the relative abundance of Streptomyces under M1 and M2 increased by 64% and 205%, by 31% and 53% for Gemmatimonas; however, under the conditions of N2 input, the relative abundance of Streptomyces under M1 and M2 increased by 10% and 93%, respectively, the Gemmatimonas for M1 reduced by 2%, and increased by 56% for M2. Moreover, the relative abundance of soil Streptomyces and Gemmatimonas was negatively related with soil N₂O emission, but positively related with maize yield. 【Conclusion】 Arbuscular mycorrhizal fungi could reduce soil N₂O emission under both higher and lower N fertilizer application rate by increasing the maize N uptake, and regulating the bacterial composition, especially increasing the relative abundance of Streptomyces and Gemmatimonas.

Effects of Different Rootstocks on Flavonoids of Vitis vinifera L. cv. Tannat Grape Fruits

HAN Xiao, YANG HangYu, CHEN WeiKai, WANG Jun, HE Fei

Scientia Agricultura Sinica. 2022, 55(10): 2013-2025. doi:10.3864/j.issn.0578-1752.2022.10.011

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【Objective】Flavonoids are important metabolites of wine grapes, which have important effects on the qualities of the grape fruits and their wines. In this study, the effects of different rootstocks on the physicochemical parameters and flavonoid substances of Tannat (Vitis vinifera L.) grapes were studied to provide the theoretical basis for the selection and utilization of rootstocks.【Method】In the present research, Tannat shoots were used as scions and were greenwood grafted on four different kinds of rootstocks, including 1103P, 101-14, SO4, and Beta. On the bases of the analysis of the basic physicochemical parameters (total soluble solid content, titratable acid, pH, and 100-berry weights) of the commercial mature grape berries of these grapevines grafted on different rootstocks, the compositions and contents of the flavonoids in the corresponding grape berries were detected by using high performance liquid chromatography-mass spectrometry (HPLC-MS), in the three vintages of 2016, 2017 and 2019.【Result】 The rootstocks had little effect on the 100-berry weights of the Tannat grapes; the contents of the total soluble solids were higher in the combination of Tannat/101-14, as well as the own-rooted Tannat; the titratable acids of the grape juice of the Tannat/101-14 and the Tannat/Beta combinations were higher than the own-rooted Tannat grapes. In the part of flavonoids, the contents of anthocyanins and flavonols in the Tannat/SO4 combination was the lowest in all of these combinations; the concentrations of anthocyanins and flavonols in the Tannat/101-14 combination and the own-rooted Tannat were higher than those of other combinations; the concentrations of flavanols in the skins of the Tannat/101-14 combination was higher. In addition, the concentrations of anthocyanins and flavonols in the Tannat/ 11103P combination was lower, but the content of flavanols in the skins of the Tannat/1103P combination was relatively high. Besides, the results of two-factor ANOVA of the year and the rootstock showed that the rootstocks had significant effects on all types of anthocyanins. All the four rootstocks showed a tendency of reducing the anthocyanins of peonidin, petunitin, malvidin, non-acylation, acetylation and coumaric acylation. In the mature fruits, the quercetins were the most abundant flavonols, followed by myricetins, while syringetins and laricitrins accounted for a smaller proportion. Rootstocks had a significant effect on the myricetins and laricitrins, which reduced the contents of myricetin and laricitrin to different degrees. Through the establishment of OPLS-DA (Orthogonal Partial Least Squares Discrimination Analysis) model, it was found that the Tannat/101-14 combination was mainly distinguished by the malvidin anthocyanin compared with the own-rooted grapes. The main difference compounds of Tannat/Beta combination were anthocyanins of malvidin, delphinidin and acetylation, the total flavanols and quercetin compounds. The difference compounds of the Tannat/SO4 combination were anthocyanins of malvidin, delphinidin, acetylation, and quercetins. The Tannat/1103P combination mainly consisted of acetylated anthocyanins and quercetins.【Conclusion】 In Beijing region, all the four rootstocks (1103P, SO4, Beta, and 101-14) showed a tendency of reducing the flavonoid concentrations, including anthocyanins of peonidin, petunitin, malvidin, non-acylation, acetylation, coumaric acylation quercetins, and laricitrins. The rootstock '101-14' was beneficial to the accumulation of anthocyanins, flavonols and flavanols in fruit skins, which was conducive to the improvement of the wine quality, so it was recommended to be used. However, Tannat grapes grafted with SO4 had less flavonoid accumulation, which was not recommended to be used.

Characterization of the Lactone Volatile Compounds in Different Types of Peach (Prunus persica L.) Fruit and Evaluations of Their Contributions to Fruit Overall Aroma

ZHANG YuanYuan,LIU WenJing,ZHANG BinBin,CAI ZhiXiang,SONG HongFeng,YU MingLiang,MA RuiJuan

Scientia Agricultura Sinica. 2022, 55(10): 2026-2037. doi:10.3864/j.issn.0578-1752.2022.10.012

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【Objective】 The objective of this study was to conduct a systematical analysis of the lactone volatile compounds in different types of ripe peach fruit (Prunus persica L.) and to evaluate the contributions of each lactone compound to peach fruit aroma. 【Method】 Multiple peach cultivars with different flesh textures, flesh colors and fruit growth periods were used in this study. The gas chromatography-mass spectrometry system was employed to identify and quantify the lactone volatile compounds in peach fruit, and the odor activity value was used to evaluate the contributions of each lactone compound to the fruit aroma of respective cultivars. 【Result】 Lactone volatile compounds were detected in ripe fruit of all peach cultivars, and a total of ten lactone volatile compounds were identified in peach fruit, including γ-hexalactone, γ-octalactone, γ-heptalactone, γ-decalactone, δ-deca-2, 4-dienolactone, δ-decalactone, γ-undecalactone, δ-octalactone, jasmine lactone, and cis-4-hydroxydodec-6-enoic acid lactone. Each lactone compound was of specific odor notes, and the lactone compounds predominantly emit fruity (reminiscent of coconut and peach), sweet, dairy, caramel, floral, and herbaceous smells. The common lactone compound shared by all cultivars was γ-hexalactone, the frequently detected lactone compounds were γ-decalactone and δ-decalactone, and some lactone compounds were specific to individual cultivars, such as cis-4-hydroxydodec-6-enoic acid lactone in Shenzhoumitao. Relatively higher numbers of lactone volatile compounds were detected in ripe fruit of melting peach cultivars, including Baihuashuimi, Shenzhoumitao, Chengxiang, Fenghuayulu (wan), and Feichenghonglidatao, while the lower numbers of lactone volatile compounds were present in stony hard peach cultivars including Xiacui, Qinwang and Huayu. The analysis of the odor activity values of the lactone volatile compounds revealed the universal contribution of γ-decalactone to the aroma of the majority of cultivars due to its low odor threshold value and high concentrations in fruit. γ-Decalactone conferred the strong characteristic peach-like odor to melting peach cultivars, including Shenzhoumitao, Chengxiang, Fenghuayulu (wan), and Baihuashuimi, while plain peach-like odor was observed in melting peach Achutao and stony hard peach Huayu due to the lower odor activity values of γ-decalactone, and the characteristic peach-like odor note was absent in stony hard peach Qinwang and Xiacui fruit as no γ-decalactone was detected. Besides, γ-octalactone contributed to the coconut and very sweet odors of specific cultivars, such as Chengxiang and Shenzhoumitao. 【Conclusion】 Lactones constituted an essential chemical group of the volatile compounds of peach fruit, and the mature peach fruit presented at least ten lactone volatile compounds. Various lactone volatile compounds and their different concentrations showed the aroma characteristics of different types of peach cultivars, especially the cultivars of different flesh textures, while not the ones of different flesh colors or fruit growth periods. γ-Hexalactone was the common lactone shared by all cultivars, γ-decalactone and δ-decalactone were frequently detected lactone compounds, and cis-4-hydroxydodec-6-enoic acid lactone and other lactones were specific to individual cultivars. γ-Decalactone, γ-octalactone and other lactones made important contributions to the characteristic peach-like odor and other unique odor notes in different peach cultivars.

Effects of Adding Quinoa Protein Pickering Emulsion on Freeze- Thaw Stability of Fish Surimi Gel

FENG Xiao,ZHANG Fan,CHEN Ying,CHENG JiaXin,CEN KaiYue,TANG XiaoZhi

Scientia Agricultura Sinica. 2022, 55(10): 2038-2046. doi:10.3864/j.issn.0578-1752.2022.10.013

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【Objective】Based on the freeze-thaw stability of quinoa stabilized Pickering emulsion, it was incorporated evenly into surimi to generate surimi gels in this study, and its feasibility to improve the freeze-thaw stability of fish protein gel was evaluated. This research aimed to prevent the deterioration of fish protein gel, caused by the temperature fluctuation during storage and transportation.【Method】Quinoa protein Pickering emulsion was prepared and distributed in surimi, followed by heating process to generate surimi gels. The surimi gels with different contents of Pickering emulsion and without emulsion were subjected to three freeze-thaw cycles, and then, the texture, color, water and ice crystal distribution and drip loss of surimi gels were measured. 【Result】The quinoa protein Pickering emulsion improved the lightness and whiteness of fish surimi gel, and inhibited the changes of color after freeze-thaw cycles. Meanwhile, the Pickering emulsion addition delayed the changes of hardness and chewiness of surimi gels during freeze-thaw cycles. It was found that quinoa protein Pickering emulsion had no effect on the moisture distribution of surimi gels before freeze-thaw cycles, but significantly increased the proportion of immobile water and decreased free water content in surimi gels after freeze-thaw cycles. Therefore, the drip loss of surimi gel was decreased by emulsion addition. Furthermore, the emulsion addition decreased the diameter of ice crystals formed in surimi gel, reduced the damage to muscle tissues, and decreased the free water content. 【Conclusion】The Quinoa protein Pickering emulsion weakened the adverse impact of freeze-thaw cycles on the color and textural properties, maintained the gel structure, and improved the freeze-thaw stability of fish surimi gel, which maintained its quality and nutritional value. Quinoa protein Pickering emulsion was promising to become an innovative antifreeze to be applied in frozen food.

The miR-221 Inhibits the Viability and Proliferation of Ovine Mammary Epithelial Cells by Targeting IRS1

KE Na,HAO ZhiYun,WANG JianQing,ZHEN HuiMin,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin,ZHAO ZhiDong,HUANG ZhaoChun,LIANG WeiWei,WANG JiQing

Scientia Agricultura Sinica. 2022, 55(10): 2047-2056. doi:10.3864/j.issn.0578-1752.2022.10.014

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【Background】MicroRNAs (miRNA) are a type of small RNAs (18-23 nt) that are widely involved in the regulation of mammogenesis and milk traits in livestock animals. In our previous research, the expression level of miR-221 in non-lactating mammary gland was found to be 3.6-time higher than in mammary gland at lactation period in Small-Tailed Han sheep by using RNA-Seq. However, the regulatory mechanism of miR-221 on ovine mammary gland development is still unclear. 【Objective】The aim of this study was to investigate the inhibition of miR-221 on the viability and proliferation of ovine mammary epithelial cells by targeting insulin receptor substrate 1 (IRS1) gene, so as to provide a theoretical reference for revealing the molecular regulation mechanism of miR-221 on ovine lactation performance.【Method】In the study, mammary gland, heart, liver, kidney, spleen, lung, Longissimus dorsi muscle and ovary tissues were collected in Small-Tailed Han sheep, and the expression profiles of miR-221 were constructed in ovine eight tissues by using reverse transcription-quantitative PCR (RT-qPCR). The effects of miR-221 on the viability and proliferation of ovine mammary epithelial cells (OMECs) were investigated by using cell transfection, CCK-8 and Edu assays. The miRDB and miRanda were used to predict the target genes of miR-221. Based on functional enrichment analysis, an investigated target gene was screened. The target relationship between miR-221 and the predicted target gene was investigated by constructing wild-type and mutant-type report vectors for the target gene by using dual luciferase reporter assay. Finally, the effects of over-expressed and silenced miR-221 on expression levels of the target gene and other functional genes in downstream signaling pathways were detected.【Result】The miR-221 was expressed in ovine eight tissues including mammary glands, with the highest expression levels in lung and spleen, and the lowest expression levels in Longissimus dorsi muscle and kidney. The CCK-8 assay result revealed that miR-221 mimic inhibited the viability of OMECs, whereas miR-221 inhibitor promoted the viability of OMECs. The Edu result found that miR-221 mimic reduced the number of Edu-labeled positive OMECs. On the contrary, miR-221 inhibitor increased the number of Edu-labeled positive OMECs. The result from dual luciferase reporter assays showed that the miR-221 mimics reduced the luciferase activity of the 3′UTR region of IRS1, while miR-221 inhibitor increased the luciferase activity. This suggested that IRS1 was a target gene of miR-221. The results from RT-qPCR further found that over-expressed miR-221 reduced expression levels of IRS1 and PIK3R1 in OMECs (P<0.05), while silenced miR-221 enhanced the levels of the two genes in expression (P<0.05). No effect on IGF1R was found for over-expressed and silenced miR-221 in OMECs (P>0.05).【Conclusion】The miR-221 inhibited the viability and proliferation of OMECs by reducing IRS1 expression.

LKB1 Regulates Steroids Synthesis Related Genes Expression in Bovine Granulosa Cells

ZHANG Jing,ZHANG JiYue,YUE YongQi,ZHAO Dan,FAN YiLing,MA Yan,XIONG Yan,XIONG XianRong,ZI XiangDong,LI Jian,YANG LiXue

Scientia Agricultura Sinica. 2022, 55(10): 2057-2066. doi:10.3864/j.issn.0578-1752.2022.10.015

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【Background】 The Steroids synthesis capacity of ovarian granulosa cells plays the important roles in the development and maturation of follicles, however, the key regulators were involved in this process remains largely unknown. Our previously research reported that Liver kinase B1 (LKB1) influenced the cellular lipid metabolism, which is close associated with steroids synthesis. Further, another study showed that knockout of LKB1 caused premature ovarian failure in mice. 【Objective】 The aim of this study was to study the expression pattern of LKB1 in bovine follicle and its regulation on steroid synthesis related genes expression in granulosa cells (GCs),and provided a theoretical basis for the research of the reproductive physiological regulation in the cow.【Method】The expression pattern of LKB1 in follicle was detected by immunohistochemically assay. Then the primary follicular granulosa cells were isolated and identified by immunofluorescence staining incubated by follicle stimulating hormone receptor (FSHR) antibody. Next, these verified granulosa cells were used as the cell model. On one hand, LKB1 loss-of-function was mediated by siRNAs. qRT-PCR was performed to measure LKB1 regulation of steroid hormone synthesis related genes expression. On the other hand, LKB1 gain-of-function was mediated by adenovirus. qRT-PCR and ELISA analysis were carried out to confirm the changes of above detected genes influenced by LKB1 and estradiol (E2) secretion, respectively. 【Result】 The data showed that: 1) LKB1 protein expressed in all cell types of follicles and the positive signal in granulosa cells is significantly higher than that of theca cells, which is verified by quantitative analysis. 2) The morphology of isolated bovine follicular granulosa cells was shape of round, which were specifically labeled by follicle stimulating hormone receptor (FSHR) using immunofluorescence staining, with 95% of positive cells. 3) The interference efficiency of LKB1 treated by siRNA1 and siRNA2 was respectively 48% (P<0.05) and 52% (P<0.05) to that of control. Knockdown of LKB1 significantly down-regulated mRNA levels of STAR (P<0.01), CYP11A1 (P<0.01) and CYP19A1 (P<0.05), with the 60%, 80% and 50% decrease to those of the control. 4) The highly infected efficiency was observed infected by LKB1-OE and control adenovirus. In contrast, overexpression of LKB1 dramatically increased mRNA levels of STAR (P<0.01), CYP11A1 (P<0.01) and CYP19A1 (P<0.05), which was associated with elevation of E2 secretion. 【Conclusion】In summary, LKB1 was highly expressed in follicular granulosa cells, which promoted the expression of steroids synthesis related genes and E2 secretion. This result provides directly theoretical evidence for the LKB1 regulation of steroids hormone synthesis in bovine.

Mechanism of NMRAL1 Regulating Influenza Virus Replication

YAN Ya,WANG GuangWen,KONG FanDi,WANG XuYuan,WANG YiHan,LI JunPing,ZHAO YuHui,LI ChengJun,CHEN HuaLan,JIANG Li

Scientia Agricultura Sinica. 2022, 55(10): 2067-2076. doi:10.3864/j.issn.0578-1752.2022.10.016

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【Objective】Influenza virus is a zoonotic pathogen that often causes a pandemic and poses a great threat to human health, and the influenza viruses are prone to variants and can constantly escape the host cell immune response and develop resistance to existing anti-influenza drugs, so the search for new ways to fight influenza is imminent. This study aimed to explore the effect of NMRAL1 (NmrA-like family domain-containing protein 1) on influenza virus replication, and to reveal the molecular mechanism by which it functioned, so as to provide a potential target for anti-influenza drugs development. 【Method】In this study, siRNA interference technology was used to down regulate the expression of NMRAL1 in A549 cells, and the expression levels of NMRAL1 were detected by Western Blot. Virus titers in cell supernatants at 24 h and 48 h after infection with two different subtypes influenza viruses, including a/Anhui/ 2/2005(AH05)(H5N1) and a/WSN/33(H1N1), were detected using the plaque assay. To determine the specific stage at which NMRAL1 affected influenza virus replication, NMRAL1 was overexpressed by transiently transfecting NMRAL1-Myc-pCAGGS plasmid in HEK293T cells, and the effect of overexpressing NMRAL1 on influenza virus polymerase activity was examined by luciferase reporter system. The influenza virus NP protein was stained by using immunofluorescence, and the down-regulated expression of NMRAL1 on the localization of NP protein at 3, 4, 5, 6 and 8 h post infection was assessed respectively by confocal assay to determine whether down-regulated expression of NMRAL1 affected the process of influenza virus vRNP import and export. Western Blot was used to detect the effect of NMRAL1 knockdown on the expression of viral proteins and on the expression of IFN stimulated genes (ISGs) downstream of type I interferon pathway activated by influenza virus. Indirect immunofluorescence assay was utilized to further verify the effect of NMRAL1 on influenza virus replication. 【Result】Western Blot assay showed that NMRAL1 siRNA could significantly down regulate NMRAL1 expression in A549 cells. With the down-regulated expression of NMRAL1, A549 cells were infected with H5N1 and H1N1 viruses, respectively. Then the virus titers in the cell supernatant were measured by plaque assay, which showed that the virus titers in the supernatant of cells at 24 and 48 h after infection with H5N1 or H1N1 were significantly decreased, meaning that NMRAL1 could promote the replication of different subtypes influenza viruses. To further explore the specific mechanism by which NMRAL1 regulated influenza virus replication, a luciferase reporter system was used to detect influenza virus polymerase activity, and it was found that the overexpression of NMRAL1 had no effect on influenza virus polymerase activity. The results of confocal assay showed that the down-regulated expression of NMRAL1 did not affect the process of NP nuclear import and export, meanwhile Western Blot assay indicated that down-regulated expression of NMRAL1 did not affect the expression of each viral protein. However, the results of the fluorescence quantitative PCR assay showed that down-regulated expression of NMRAL1 was able to promote the up-regulation of IFN-β mRNA levels induced by influenza virus infection, and Western Blot assay found that down expression of NMRAL1 promoted the expression of MxA and IFITM3 antiviral proteins downstream of type I interferon pathway. Meanwhile, the indirect immunofluorescence assay showed that the down expression of NMRAL1 could significantly inhibit influenza virus replication. 【Conclusion】 Those results demonstrated that, during influenza virus infection, NMRAL1 did not affect the process of influenza virus invasion as well as transcription translation, but rather inhibited the expression of antiviral factors, such as MxA and IFITM3, by inhibiting type I interferon pathway activation, which ultimately promoted influenza virus replication. This study confirmed that the host factor NMRAL1 positively regulated influenza virus replication and enriched the network of host factors involved in influenza virus replication.

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